This invention relates to the molecular biology and virology of the hepatitis C virus (HCV). More specifically, this invention relates to (1) carboxy terminus fragments of the HCV NS3 protein having helicase activity and improved solubility in extraction and assay buffers, (2) methods of expressing the novel NS3 protein fragments having helicase activity and improved solubility (3) recombinant NS3 protein fragments having helicase activity and improved solubility; (4) NS3 protein mutant fragments; and (5) method of using the HCV NS3 protein fragments for screening helicase inhibitors as potential therapeutic agents.
Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be virally induced, and is distinguished from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that there may be three types of NANBH: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of causative agents is unknown. Recently, however, a new viral species, hepatitis C virus (HCV) has been identified as the primary (if not only) cause of blood-associated NANBH (BB-NANBH). See for example, PCT WO89/04669 and U.S. patent application Ser. No. 7/456,637, filed Dec. 21, 1989, now abandoned, incorporated herein by reference. Hepatitis C appears to be a major form of transfusion-associated hepatitis in a number of countries, including the United States and Japan. There is also evidence implicating HCV in induction of hepatocellular carcinoma. Thus, a need exists for an effective method of treating HCV infection: currently, there is none.
HCV is a positive strand RNA virus. Upon infection, its genomic RNA produces a large polyprotein that is processed by viral and cellular proteins into at least 10 different viral proteins. Like other positive strand RNA viruses, replication of the positive strand involves initial synthesis of a negative strand RNA. This negative strand RNA, which is a replication intermediate, serves as a template for the production of progeny genomic RNA. This process is believed to be carried out by two or more viral encoded enzymes, including RNA dependent RNA polymerase and RNA helicase. RNA polymerase copies template RNA for the production of progeny RNA. This enzyme does not synthesize RNA molecules from DNA template.
The RNA helicase unwinds the secondary structure present in the single-strand RNA molecule. The helicase also unwinds the duplex RNA into single-strand forms. Genomic HCV RNA molecules contain extensive secondary structure. Replication intermediates of HCV RNA are believed to be present as duplex RNA consisting of positive and negative strand RNA molecules. The activity of RNA helicase is believed to be crucial to RNA dependent RNA polymerase which requires unwound single stranded RNA molecules as a template. Therefore, the biological activity of helicase is believed to be required for HCV replication.
NS3 proteins of the three genera of the Flaviviridae family: flavivirus, pestivirus and HCV, have been shown to have conserved sequence motifs of a serine-type proteinase and of a nucleoside triphosphatase (NTPase)/RNA helicase. One third of the Nxe2x80x2-terminal of the HCV NS3 protein has been shown to be a trypsin like serine proteinase which cleaves the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. Faila et al., J. Virol. 68:3753-3760 (1994). Two thirds of the NS3 Cxe2x80x2-terminal fragment has been shown to encode NTPase/RNA helicase activity. Choo et al., PNAS, 88:2451-2455 (1991) and Gorbalenya et al., Nucleic Acids Res., 17:4713-4729 (1989). Suzich et al. showed that two thirds of the carboxy terminal fragment of HCV NS3 expressed in E. coli had polynucleotide-stimulated NTPase activity. J. Virol, 67:6152-6158 (1993). Gwack et al., in xe2x80x9cNTPase Activity of Hepatitis C Virus NS3 Protein Expresed in Insect Cellsxe2x80x9d Mol. Cells. 5(2): 171-175 (1995), showed two HCV NS3 proteins, p70 and p43, were expressed in a baculovirus expression system. The p70 showed a specific NTPase activity that was inhibited by NS3 monoclonal antibodies. Warrener et al., xe2x80x9cPestivirus NS3 (p80) Protein Possesses RNA Helicase Activity,xe2x80x9d J. Virol. 69:1720-1726 (1995), demonstrated that bovine viral diarrhea virus (BVDV) NS3 protein expressed in a baculovius expression system had a RNA helicase activity. JP 0631 9583A descibes the preparation of a helicase protein encoded by HCV by introducing a HCV helicase gene into the non-essential region of a baculovirus. The helicase amino acid sequence is reported as 1200 through 1500 of the HCV polyprotein. All documents mentioned above arm incorporated herein in their entirety by reference.
We have now invented recombinant HCV NS3 protein fragments having helicase activity and improved solubility, fusion HCV NS3 protein fragments having helicase activity and improved solubility, truncated and altered HCV NS3 protein fragments having helicase activity and improved solubility, and cloning and expression vectors therefore, and methods for using these protein fragments in screening assays to assess whether a compound is capable of inhibiting RNA helicase activity and thus inhibiting HCV replication.
FIG. 1 shows the sequence of the HCV-1 NS3 protein (SEQ ID NO:1 ), which is approximately from amino acid 1027 to 1657 of the HCV-1 polyprotein (SEQ ID NO:6).
FIG. 2 is a schematic presentation of the HCV NS3 protein. The numbers indicate the amino acid positions of the HCV-1 polyprotein.
FIG. 3 shows the conserved sequence motif of DEXH box RNA helicase proteins (A) and comparative alignment of the RNA helicase domain of the HCV NS3 protein (A). The numbers between boxes indicate the distance in amino acids residues.
FIG. 4 shows the structure of double strand RNA substrate for RNA helicase assay. The thick line indicates the 32P-labeled RNA strand. The thin line indicates the unlabeled RNA strand.
FIG. 5 shows the expression and purification of HCV NS3 from E. Coli. M: protein size markers, Lane 1: Total protein from uninduced cells, Lane 2: Total protein from 3 hr IPTG induced cells, Lane 3: HCV NS3:His-tag fusion protein purified by nickel binding chromatography.
FIG. 6 shows the results of an RNA helicase assay of the HCV NS3 protein fragments. Lane 1; Fraction from negative control cell (pET vector only), Lane 2:3 mM Mn2+, Lane 3: no Mn2+, Lane 4:3 mM Mg2+, Lane 5:no Mg2+, Lane 6:3 mM KCl , Lane 7:no ATP, Lane 8:1 mM ATP, Lane 9:preincubation of the NS3 protein with NS3-specific monoclonal antibody, Lanes 10, 11: preincubation of the NS3 protein with anticonnexin monoclonal antibody at 0.5, xcexcg, 1.0 xcexcg per 20xcexcl, respectively. Monoclonal antibodies were preincubated with the S3 protein at room temperature for 5 min.
FIGS. 7A-B shows the activity profiles of the HCV NS3 RNA fragment having helicase activity with different ATP and divalent cations concentrations. The effects of cations were tested at two different ATP concentrations (1 mM and 5 mM).